A large body of evidence suggests that aging of primary cell lines is accompanied by the accumulation of defective enzyme molecules. Although the mechanism by which this occurs is not clear, it seems plausible that the resulting metabolic imbalance is a major cause of cellular senescence. Since it is well known that allosteric properties of enzymes are more sensitive to structural changes than catalytic properties, we plan to investigate the regulatory properties of selected enzymes as a function of passage level in human diploid fibroblasts. The enzymes to be studied include the early enzymes in purine and pyrimidine biosynthesis, important control sites in the synthesis of nucleic acid precursors, as well as phosphofructokinase, a key regulatory enzyme in glycolysis. In order to avoid alterations in regulatory behavior which often accompanies enzyme isolation and which would make comparison difficult, we will use a novel approach which involves studying the enzymes in intact cells made permeable to small molecules by the treatment with dextran sulfate. This permeable cell system will permit the study of enzymes at physiological concentrations in their natural macromolecular environment. The allosteric properties observed under these conditions should be a more accurate reflection of enzyme regulation in vivo. Any changes observed in these properties during the aging process should therefore be of special relevance to the changes in cell physiology that accompany aging.